Project Summary Neurons are organized within cellular circuits which give rise to all brain activity. An anatomical understanding of neuronal architecture is crucial to figuring out how the brain processes information. Commonly, the visualization of neurons has relied on labeling with different colored fluorescent proteins in order to distinguish one from another, however a major limitation of this approach is the low diversity of colors that are available. Because neurons can project over long distance and along complex paths, many overlapping cells will be indistinguishable from one another when a small color palette is used. We propose technology which decouples labelling diversity from the limitation of dye colors. We propose labeling each cell with a combination of peptides rather than a single dye and performing iterative immunofluorescence staining which can re-use colors. Using this technology, we will create over one million discernable labels which will be used to map the location of an unprecedented number of cells within brain tissue. We will perform proof of concept studies on cell lines using standard plasmid preparation and transfection techniques (Aim 1). Next we will pursue two viral strategies to genetically label neurons (Aim 2). We will encode peptide combinations within replication-deficient Sindbis vectors which can infect neurons rapidly and induce high levels of peptide expression and also engineer adeno associated viruses (AAV) to deliver labels because AAV can be administered systemically and selectively target neuronal subtypes. Together, these tools will be used to map, in three dimensions, an entire viral injection volume (Aim 3). It is expected that the technology developed from this proposal will have a drastic impact on cellular labelling and brain connectomics.